CN113341000B - Method for determining concentration of sodium danshensu and prunasin in dog plasma - Google Patents
Method for determining concentration of sodium danshensu and prunasin in dog plasma Download PDFInfo
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Abstract
The invention discloses a method for determining the concentration of Sodium Danshensu and Prunasin in dog plasma by an HPLC-MS/MS method.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a method for determining the concentrations of Sodium Danshessu and Prunasin in canine plasma by an HPLC-MS/MS method.
Background
Hepatic fibrosis is one of the liver diseases. After the human body suffers from hepatic fibrosis, symptoms such as fatigue, weakness, anorexia and the like are easy to appear, and symptoms such as chronic dyspepsia, bleeding and the like are possibly also appeared, and the symptoms can cause great harm to the human body.
The tablet for strengthening body resistance and removing blood stasis is one of the commonly used Chinese patent medicines for treating hepatic fibrosis, and comprises the following main components: schisandrin (Schizandrin), schisandrin B (gamma-Schizandrin), maosolide (Massonia Lactone), danshensu Sodium (Sodium Danshensu), prunasin (Prunnasin), amygdalin (Amygdalin), naringenin (Naringenin), quercetin (Quercetin), rutin (Rutin), phytolaccin (Ombusoside) and its metabolite phytolaccin (Ombuin), quercitrin (Quercin), kaempferol (Kaempferol), and shorea Sodium enoate (Sodium 5-Hydroxy-2-Decenoic). The toxicity kinetics characteristics of the tablet for strengthening the body resistance and removing blood stasis in the plasma of Beagle dogs (Beagle dogs) are detected, the exposure level of the main components of the medicine in Beagle dogs is evaluated, and the method has important significance for helping to know the action and effect of the medicine in human bodies. Therefore, those skilled in the art are continuously working on the HPLC-MS/MS method for determining the concentration of the main component of the tablet for strengthening body resistance and removing blood stasis in plasma.
Disclosure of Invention
In order to overcome the defects in the prior art, the embodiment of the invention provides a method for determining the concentrations of Sodium Danshensu and Prunasin in canine plasma by an HPLC-MS/MS method, and the working conditions of the HPLC-MS/MS analysis and determination process are as follows:
(1) And PHLC conditions:
a chromatographic column: inertStain AQ-C18 (2.1mm. Times.50mm, 5.0 μm), SHIMADZU;
column temperature: 35 ℃;
mobile phase A:0.1% acetic acid-water solution;
mobile phase B: methanol-acetonitrile solution (7/3, v/v);
sample introduction volume: 3.00 mu L;
operating time: 4.80min;
retention time: sodium Danshensu, about 1.68min; prunasin, about 1.82min; osamide, about 2.04min;
(2) Mass spectrum conditions:
mass spectrometry: AB SCIEX API5000 LC/MS/MS system;
an ion source: ESI electrospray ionization;
ionization mode: negative;
vortex ion spray temperature: at 550 ℃.
Further, the method comprises the following steps:
(1) 1.00mg/mL of each of Sodium Danshensu, prunasin and Osalmide is taken and respectively dissolved in methanol to prepare Sodium Danshensu stock solution, prunasin stock solution and Osalmide stock solution, and the obtained solution is sealed at the temperature of less than or equal to-15 ℃ and stored in a shading way, wherein Osalmide is taken as an internal standard;
diluting the Sodium Danshensu stock solution and the Prunasin stock solution with methanol solution to a concentration of 2 x 10 respectively 5 、1.6*10 5 The calibration of ng/mL indicates the sample working solution; mixing the Sodium Danshensu and the Prunasin with equal concentration, and diluting with methanol solution to obtain a mixture with a concentration of 4 x 10 4 、1.6*10 4 1600, 400 and 200ng/mL of calibration marking sample working solution;
diluting the Sodium Danshensu stock solution and the Prunasin stock solution with methanol solution to a concentration of 1.5 x 10 respectively 5 、1.0*10 5 ng/mL quality control working solution; mixing the Sodium Danshensu and the Prunasin and diluting to a concentration of 6 x 10 with methanol solution 4 600 and 200ng/mL quality control working solution;
diluting the Osalmide stock solution with methanol solution to a concentration of 1 x 10 4 50ng/mL internal standard working solution;
diluting the calibration marker sample working solution to a concentration of 1 × 10 with a blank matrix 4 8000, 2000, 800, 80, 20, 10ng/mL of said calibration marker, said blank matrixCanine plasma, which is free of analyte and internal standard, in this example refers to plasma free of Sodium Danshensu, prunasin, and osamide;
diluting the quality control working solution with the blank matrix into the quality control samples with the concentrations of 7500, 5000, 300, 30 and 10 ng/mL;
(2) Sample treatment: and (3) taking the calibration marker sample and the quality control sample with the same volume, respectively adding 270 mu L of 50ng/mL internal standard working solution, closing a plate, performing vortex mixing, centrifuging, taking 150 mu L of supernatant to the 96-well plate, and adding 100 mu L of methanol solution. Then, the pore plate is sealed, low-speed vortex mixing is carried out, and 3.0 mu L of the mixture is taken out and analyzed on a high performance liquid chromatography mass spectrometer (HPLC-MS/MS);
(3) Preparation of a standard curve: making a standard curve by using Y = aX + b, wherein X is the concentration of the analyte, and Y is the area ratio of chromatographic peaks;
(4) Quantitative analysis: and (3) processing the sample to be detected according to the step (2), and calculating the concentrations of Sodium Danshessu and Prunasin in the sample to be detected according to the standard curve obtained in the step (3).
Further, the centrifugation conditions in the step (2) are as follows: centrifuge at 4000rpm for 10min at 10 ℃.
Further, the step (2) further comprises processing a Blank (BK) sample: sucking 30 mu L of blank matrix, adding 270 mu L of methanol solution, sealing the plate, then carrying out vortex mixing, centrifuging at 10 ℃ and 4000rpm for 10min, taking 150 mu L of supernatant after centrifugation, adding 100 mu L of methanol solution, then sealing the plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
Further, the step (2) also comprises the following steps of processing the QC0 samples: sucking 30 mu L of blank matrix, adding 270 mu L of the internal standard working solution, sealing the plate, performing vortex mixing, centrifuging at 10 ℃ and 4000rpm for 10min, taking 150 mu L of supernatant into the 96-well plate after centrifugation, adding 100 mu L of methanol solution, sealing the well plate, performing low-speed vortex mixing, and taking 3.0 mu L for analysis.
Further, the step (2) also comprises the processing of the ULOQ without IS sample: adding 270 mu L of methanol solution into a blank substrate without an internal standard at the highest quantitative limit, sealing the plate, then carrying out vortex mixing, centrifuging for 10min at the temperature of 10 ℃ and the speed of 4000rpm, taking 150 mu L of supernatant after centrifugation, putting the supernatant into the 96-well plate, and adding 100 mu L of methanol solution. The well plate was then sealed and mixed by low speed vortex and 3.0. Mu.L was taken for analysis.
Further, the step (2) also comprises the treatment of the matrix effect sample: taking 30 mu L blank matrix of a single donor (n is more than or equal to 6), adding 270 mu L methanol solution, carrying out vortex centrifugation, taking 150 mu L supernatant, adding 100 mu L Neat solution, sealing the pore plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
Further, the step (2) further comprises the following steps of: taking a blank matrix of a single donor, taking 30 mu L of the blank matrix mixed by a plurality of donors to form the recovery rate sample, adding 270 mu L of methanol solution, carrying out vortex centrifugation, taking 150 mu L of supernatant, adding 100 mu L of liquid, sealing the pore plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
The invention has the following beneficial effects: through optimization of technological conditions such as chromatographic columns, dissolving solution, mobile phase, retention time and the like, the whole testing process is more feasible, the measurement result is increased accurately, residues are reduced, the accuracy is improved, the recovery rate is high, and the matrix effect and the interference are reduced.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 is a spectrum of a Sodium Danshensu-Reagent sample in an example of the present invention;
FIG. 2 is a spectrum of a Blank sample of Sodium Danshensu-Blank in an example of the present invention;
FIG. 3 is a spectrum of a Sodium Danshensu-QC0 sample in an example of the present invention;
FIG. 4 is a spectrum of a Sodium Danshensu-Carryover Blank sample in an example of the present invention;
FIG. 5 is a spectrum of a Sodium Danshensu-LLOQ sample in an example of the present invention;
FIG. 6 is a spectrum of a Sodium Danshensu-ULOQ sample in an example of the present invention;
FIG. 7 is a spectrum of a Prunasin-Reagent blank sample in an example of the present invention;
FIG. 8 is a spectrum of a Prunasin-Blank sample in an example of the present invention;
FIG. 9 is a spectrum of a Prunasin-QC0 sample in an example of the invention;
FIG. 10 is a spectrum of a sample of Prunasin-Carryover Blank in an example of the present invention;
FIG. 11 is a spectrum of a Prunnasin-LLOQ sample in the example of the present invention;
FIG. 12 is a spectrum of a sample of Prunasin-ULOQ in an example of the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "upper", "lower", "bottom", "inner", "outer", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate a number of the indicated technical features. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature.
In order to achieve the above objects, the present invention provides a method for determining the concentrations of Sodium Danshessu and Prunasin in canine plasma by HPLC-MS/MS method, comprising the following steps.
1. Working condition of HPLC-MS/MS analysis determination process
(1) And HPLC conditions are as follows:
a chromatographic column: inertStain AQ-C18 (2.1mm. Times.50mm, 5.0 μm), SHIMADZU;
column temperature: 35 ℃;
a mobile phase A:0.1% acetic acid-water solution;
and (3) mobile phase B: methanol-acetonitrile solution (7/3, v/v);
pump gradient:
sample introduction volume: 3.00 mu L;
operating time: 4.80min;
retention time: sodium Danshensu, about 1.68min; prunasin, about 1.82min; osamide, about 2.04min;
(2) Mass spectrum conditions:
mass spectrometry: AB SCIEX API5000 LC/MS/MS system;
an ion source: ESI electrospray ionization;
ionization mode: negative;
vortex ion spray temperature: 550 ℃;
2. sample configuration
Preparation of stock solution: 1.00mg/mL of Sodium Danshensu, prunasin and Osalmide are taken respectively and dissolved in methanol to prepare Sodium Danshensu stock solution, prunasin stock solution and Osalmide stock solution, the mixture is sealed at the temperature of less than or equal to-15 ℃ and stored in a shading way, wherein Osalmide is taken as an internal standard; as shown in table 1.
Preparation of calibration marking sample working solution: diluting the Sodium Danshensu stock solution and the Prunasin stock solution with methanol solution to a concentration of 2 x 10 respectively 5 、1.6*10 5 The ng/mL of calibration label sample working solution; mixing the Sodium Danshensu and the Prunasin with equal concentration, and diluting with methanol solution to obtain a mixture with a concentration of 4 x 10 4 、1.6*10 4 1600, 400 and 200ng/mL of calibration marking sample working solution; as shown in table 2.
Preparing a quality control working solution: diluting the Sodium Danshensu stock solution and the Prunasin stock solution with methanol solution to a concentration of 1.5 x 10 respectively 5 、1.0*10 5 ng/mL quality control working solution; mixing the Sodium Danshensu and the Prunasin and diluting to a concentration of 6 x 10 with methanol solution 4 600, 200ng/mL quality control working solution; as shown in table 3.
Preparing an internal standard working solution: diluting the Osalmide stock solution with methanol solution to a concentration of 1 x 10 4 50ng/mL internal standard working solution; as shown in table 4.
Preparation of neat solution: diluting the quality control solution and the internal standard working solution into a neat solution by using a methanol solution; as shown in table 5.
Note: the calibration marking sample working solution, the quality control working solution, the internal standard working solution and the pure solution with the recovery rate and the matrix effect can be changed in the dilution process/volume as long as the final concentration is unchanged. As long as the solution ID is unique, the solution ID can be changed without affecting the traceability of the experiment. And were all prepared at room temperature.
Calibration of the arrangement of the markers: diluting the calibration marker sample working solution to a concentration of 1 x 10 with a blank matrix 4 8000, 2000, 800, 80, 20, 10ng/mL of the calibrator, the blank matrix being canine plasma without analyte and internal standard, in this example Sodium Danshessu, prunnasin and Osalmide; as shown in table 6.
Preparation of quality control samples: diluting the quality control working solution with the blank matrix into the quality control samples with the concentrations of 7500, 5000, 300, 30 and 10 ng/mL; as shown in table 7.
3. Sample processing
And (3) taking a 96-well plate, and labeling a calibration marking sample, a quality control sample, a blank sample, a QC0 sample, a ULOQ without IS sample, a Reagent sample, a matrix effect sample and a recovery rate sample on the 96-well plate to form a sample map.
Sucking 30 mu L of the calibration marker sample and the quality control sample and respectively putting the calibration marker sample and the quality control sample into a calibration marker sample hole and a quality control sample hole;
sucking 30 mu L of blank matrix into a blank sample hole, wherein the blank sample has no analyte and no internal standard;
sucking 30 mu L of blank matrix and placing the blank matrix into a QC0 sample hole;
sucking 30 μ L of blank matrix without internal standard at the highest limit of quantitation and placing the blank matrix into the ULOQ without IS sample hole;
pipette 30.0 μ L of aqueous solution into the wells of the Reagent sample in the 96-well plate.
Sucking 30 mu L blank matrix of a single donor (n is more than or equal to 6) and placing the blank matrix into a matrix effect sample hole;
a single donor blank was removed and 30. Mu.L of the pooled blank from multiple donors was pipetted into the recovery sample well.
And respectively adding 270 mu L of the internal standard working solution into the calibration marker sample, the quality control sample and the QC0 sample, carrying out vortex mixing after closing a plate, and centrifuging for 10min at the temperature of 10 ℃ and the rpm of 4000. After centrifugation, 150. Mu.L of the supernatant was transferred to the 96-well plate, and 100. Mu.L of methanol solution was added. The well plate was then sealed and mixed by low speed vortex and 3.0. Mu.L was taken for analysis.
And adding 270 mu L of methanol solution into the blank sample, the ULOQ without IS sample and the Reagent sample, sealing the plates, performing vortex mixing, and centrifuging for 10min at 10 ℃ and 4000 rpm. After centrifugation, 150. Mu.L of the supernatant was transferred to the 96-well plate, and 100. Mu.L of methanol solution was added. The well plate was then sealed and mixed by low speed vortexing and 3.0 μ L was taken for analysis.
Adding 270 mu L of methanol solution into the matrix effect sample and the recovery rate sample, sealing a plate, then whirling and mixing uniformly, and centrifuging for 10min at 10 ℃ and 4000 rpm. After centrifugation, 150. Mu.L of the supernatant was applied to the 96-well plate, and 100. Mu.L of the Neat solution was added. The well plate was then sealed and mixed by low speed vortexing and 3.0 μ L was taken for analysis.
4. Establishment of linear regression model
The calibration marker and the quality control sample are detected according to the chromatographic and mass spectrum conditions, and the chromatogram acquisition and the chromatographic peak integration are processed by software analysis 1.6.3 and applied Biosystem. The chromatographic peak area ratio of the Sodium Danshensu internal standard Osalmide and the Prunasin internal standard Osalmide is taken as the ordinate, the concentration of Sodium Danshensu and Prunasin in the canine plasma is taken as the abscissa, and the weight is used for weighting (the weight is 1/X) 2 ) The least square method is used for carrying out linear regression by using the concentration (X) of Sodium Danshensu and Prunasin in canine plasma and the area ratio (Y) of chromatographic peaks, and the constructed linear regression equation (Y = aX + b) is a standard curve. The results show that the calibration curves of Sodium Danshensu and Prunasin range from 10.0ng/mL to 10000 ng/mL, and the standard curves are made well linearly.
5. Quantitative analysis
And (3) treating the test sample according to the method of the sample treatment step, and calculating according to the standard curve equation obtained in the sample treatment step to obtain the concentration of the analytes Sodium Danshensu and Prunasin in the sample to be detected.
6. Evaluation of test methods
The Blank (BK) sample, QC0 sample, and ULOQ with IS samples were used to correct each other to reduce interference.
The matrix effect sample and the recovery sample are used to evaluate the matrix effect and the recovery of the method. The analysis shows that the matrix effect of the method is 100-110%. The analytical SodiumDanshensu recovery rate of the process was: 72.5% -100%, and the recovery rate of Prunasin is as follows: 90 to 100 percent.
The principle and the implementation mode of the invention are explained by applying specific embodiments in the invention, and the description of the embodiments is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (7)
1. A method for determining the concentration of danshensu sodium and prunasin in dog plasma by HPLC-MS/MS method is characterized in that the working conditions of the HPLC-MS/MS analysis and determination process
(1) And HPLC conditions are as follows:
and (3) chromatographic column: inertSustain AQ-C18,2.1mm x 50mm,5.0 μm, SHIMADZU;
column temperature: 35 ℃;
mobile phase A:0.1% acetic acid-water solution;
mobile phase B: a methanol-acetonitrile solution with a volume ratio of 7;
preparation of stock solution: respectively taking 1.00mg/mL of danshensu sodium, prunasin and lauseterol, and respectively dissolving in methanol to obtain a danshensu sodium stock solution, a prunasin stock solution and a lauseterol stock solution, sealing at a temperature of less than or equal to-15 ℃, and storing in a shade way, wherein lauseterol is used as an internal standard;
pump gradient:
sample injection volume: 3.00 mu L;
operating time: 4.80min;
retention time: sodium danshensu for about 1.68min; prunasin, about 1.82min; salidrosol, about 2.04min;
(2) Mass spectrum conditions:
mass spectrometry: AB SCIEX API5000 LC/MS/MS system;
an ion source: ESI electrospray ionization;
ionization mode: a negative ion mode;
vortex ion spray temperature: 550 ℃;
the method comprises the following steps:
(1) Respectively taking 1.00mg/mL of salvianic acid A sodium, prunasin and salsolinol, respectively dissolving in methanol to obtain salvianic acid A sodium stock solution, prunasin stock solution and salsolinol stock solution, sealing at-15 deg.C or below, and storing in shade, wherein salsolinol is used as internal standard;
diluting the salvianic acid A sodium stock solution and the prunasin stock solution with methanol solution to concentration of 2 × 10 5 、1.6*10 5 The calibration of ng/mL indicates the sample working solution; mixing the sodium danshensu and the acteoside at equal concentration, and diluting with methanol solution to obtain a solution with concentration of 4 × 10 4 、1.6*10 4 1600, 400 and 200ng/mL of calibration marking sample working solution;
diluting the salvianic acid A sodium stock solution and the prunasin stock solution with methanol solution to concentration of 1.5 × 10 5 、1.0*10 5 ng/mL quality control working solution; mixing the sodium danshensu and the prunasin, and diluting with methanol solution to obtain a solution with concentration of 6 × 10 4 600 and 200ng/mL quality control working solution;
diluting the salsolinol stock solution with methanol solution to concentration of 1 × 10 4 50ng/mL internal standard working solution;
diluting the calibration marker sample working solution to a concentration of 1 × 10 with a blank matrix 4 8000, 2000, 800, 80, 20, 10ng/mL of the calibration marker, the blank matrix being canine plasma free of analyte and internal standard;
diluting the quality control working solution with the blank matrix into quality control samples with the concentrations of 7500, 5000, 300, 30 and 10 ng/mL;
(2) Sample treatment: taking the calibration marker sample and the quality control sample with equal volumes, respectively adding 270 mu L of 50ng/mL internal standard working solution, closing a plate, carrying out vortex mixing, centrifuging, taking 150 mu L of supernatant to a 96-well plate, and adding 100 mu L of methanol solution; then, the pore plate is sealed, low-speed vortex mixing is carried out, and 3.0 mu L of the mixture is sampled and analyzed on a high performance liquid mass spectrometer;
(3) And (3) preparing a standard curve: making a standard curve by using Y = aX + b, wherein X is the concentration of the analyte, and Y is the area ratio of chromatographic peaks;
(4) Quantitative analysis: and (3) processing the sample to be detected according to the step (2), and calculating the concentrations of the sodium danshensu and the prunasin in the sample to be detected according to the standard curve obtained in the step (3).
2. The method according to claim 1, wherein the centrifugation conditions in step (2) are: centrifuge at 4000rpm for 10min at 10 ℃.
3. The method according to claim 2, wherein the step (2) further comprises processing a blank sample: sucking 30 mu L of blank matrix, adding 270 mu L of methanol solution, sealing the plate, then carrying out vortex mixing, centrifuging at 10 ℃ and 4000rpm for 10min, taking 150 mu L of supernatant after centrifugation, adding 100 mu L of methanol solution, then sealing the plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
4. The method according to claim 3, wherein the step (2) further comprises processing a blank quality control sample: sucking 30 mu L of blank matrix, adding 270 mu L of the internal standard working solution, sealing the plate, performing vortex mixing, centrifuging at 10 ℃ and 4000rpm for 10min, taking 150 mu L of supernatant into the 96-well plate after centrifugation, adding 100 mu L of methanol solution, sealing the well plate, performing low-speed vortex mixing, and taking 3.0 mu L for analysis.
5. The method according to claim 4, wherein the step (2) further comprises processing the sample without the internal standard at the highest quantitative limit: adding 270 mu L of methanol solution into a blank substrate without an internal standard at the highest quantitative limit, sealing the blank substrate and then vortex and uniformly mixing the blank substrate and the methanol solution, centrifuging the blank substrate for 10min at the temperature of 10 ℃ and the speed of 4000rpm, taking 150 mu L of supernatant after centrifugation, putting the supernatant into the 96-well plate, and adding 100 mu L of methanol solution; the well plate was then sealed and mixed by low speed vortexing and 3.0 μ L was taken for analysis.
6. The method according to claim 5, wherein the step (2) further comprises processing the matrix effect sample: taking 30 mu L blank matrix of a single donor, adding 270 mu L methanol solution, carrying out vortex centrifugation, taking 150 mu L supernatant, adding 100 mu L recovery rate and matrix effect pure solution, sealing the pore plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
7. The method of claim 6, wherein the step (2) further comprises processing the recovery sample: taking a blank matrix of a single donor, taking 30 mu L of the blank matrix mixed by a plurality of donors to form the recovery rate sample, adding 270 mu L of methanol solution, taking 150 mu L of supernatant after vortex centrifugation, adding 100 mu L of recovery rate and matrix effect pure solution, then sealing the pore plate, carrying out low-speed vortex mixing, and taking 3.0 mu L for analysis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019114215463 | 2019-12-31 | ||
| CN201911421546.3A CN111198234A (en) | 2019-12-31 | 2019-12-31 | Method for determining the concentration of Sodium Danshensu and Prunasin in canine plasma |
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| AU2005270059A1 (en) * | 2004-07-02 | 2006-02-09 | E.I. Du Pont De Nemuors And Company | Antifungal polypeptides |
| CN101816715A (en) * | 2009-11-24 | 2010-09-01 | 云南理想药业有限公司 | Method for measuring content of Danshensu in shenshuaining capsules by utilizing HPLC method |
| CN110078778A (en) * | 2019-05-15 | 2019-08-02 | 广西壮族自治区中国科学院广西植物研究所 | A method of removing prunasin from passion fruit pericarp crude extract |
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2019
- 2019-12-31 CN CN201911421546.3A patent/CN111198234A/en not_active Withdrawn
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- 2020-12-29 CN CN202011592154.6A patent/CN113341000B/en active Active
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| AU2005270059A1 (en) * | 2004-07-02 | 2006-02-09 | E.I. Du Pont De Nemuors And Company | Antifungal polypeptides |
| CN101816715A (en) * | 2009-11-24 | 2010-09-01 | 云南理想药业有限公司 | Method for measuring content of Danshensu in shenshuaining capsules by utilizing HPLC method |
| CN110078778A (en) * | 2019-05-15 | 2019-08-02 | 广西壮族自治区中国科学院广西植物研究所 | A method of removing prunasin from passion fruit pericarp crude extract |
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| CN113341000A (en) | 2021-09-03 |
| CN111198234A (en) | 2020-05-26 |
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